Note: Descriptions are shown in the official language in which they were submitted.
WO96/08213 ~ PCT~S95/11395
THREE-DIMENSIONAL HUMAN CELL CULTURES ON
CARDIAC VA~VE FRAME~ORRS ~ND THEIR USES
1. INTRODUCTION
The invention relates to growing in vitro, human
cells such as fibroblasts on a three-dimensional
scaffold, comprising porcine aortic leaflets and walls,
intact heart valves, other biological scaffolding
suitable for reconstructing a valve or valve components,
for example, including but not limited to the pericardium
or the small intestinal submucosa, and biodegradable
frameworks, such that the scaffold is populated with
viable human cells having normal function, and
fibroblasts are stimulated to produce human matrix
proteins to supplement and replace the existing matrix on
the scaffold.
The resulting three-dimensional tissue constructs
have a variety of applications ranging from
transplantation or implantation in vivo for replacement
and/or reconstruction of a single valve component or the
entire heart valve, to screening cytotoxic compounds and
pharmaceutical compounds in vitro.
2. BACRGRO~ND OF THE lNV~N'l'lON
Valve replacement surgical therapy is required for
.the treatment of various types of valvular heart
diseases, including, but not limited to, aortic stenosis,
aortic regurgitation, mitral stenosis, mitral
regurgitation, pulmonary valve disease, tricuspid valve
disease, multivalvular disease, Marfan syndrome and
artificial valve disease. Two general types of valve
replacement are available: the artificial, mechanical
prosthesis or valve, and tissue biological prosthesis or
valve. There are several kinds of mechanical prosthesis,
such as the ball valve, the tilting disk and the central
flow disk. There are also several tissue prostheses,
WO96/08213 PCT~S95111395
including preserved homografts and stent-mounted, porcine
valve heterografts.
The primary advantage of the mechanical prosthesis
is durability, whereas the disadvantage is a requirement
that patients be on an anticoagulant therapy to reduce
the risk of thromboembolic complications. This is
because artificial mechanical heart valves are prone to
occlusions by thrombus, and are subject to mechanical
failures. Thromboembolism and anticoagulated hemorrhage
are still the frequent causes for reoperation and patient
death. Moreover, mechanical failure can occur suddenly
and without warning resulting in emergency surgical
interventions for replacement of the device. The
advantage of the biological prothesis is a lower risk of
thromboembolic complications, but may still require
anticoagulant therapy in some situations. Moreover, the
biological grafts which are currently used are not prone
to sudden failure. However, the biological tissue grafts
are also limited for a number of reasons. Allogeneic
human valves (derived from a donor of the same species)
are limited by the supply of donated human hearts and
recipients must undergo immunosuppressive therapy to
avoid rejection. While xenogeneic valves (derived from a
donor of a different species) are more plentiful, they
require treatment that results in calcification and a
gradual degradation over time, requiring replacement.
For example, allogeneic, transplanted heart valves may be
obtained fresh, or may be cryopreserved to maintain
viability of cellular components. Patients receiving
allogeneic transplants usually must undergo
immunosuppressive therapy. Despite such therapy, many of
the transplants become inflamed and fail within five to
ten years. Moreover, allogeneic valves are not as
readily available as the xenogeneic valves. Xenogeneic
biological valves, usually porcine or bovine in origin,
have the advantage of being identical in design and
structure to those valves being replaced, but are fixed
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WO96/08213 PCT~S95/11395
with glutaraldehyde and, therefore, are non-living. The
glutaraldehyde-treated tissues calcify over time and do
not allow infiltration and colonization by host cells,
which is necessary for remodeling. Consequently, these
xenogeneic valves degrade with time and eventually
malfunction.
Autologous human tissue (i.e., derived from the
recipient) is used for coronary and peripheral bypass
procedures, third degree burns and reconstructive
procedures involving bones and cartilage grafting. Such
use eliminates complications of immunorejection resulting
in better graft survival. Unfortunately, complications
ensue with autologous heart valve transplants e.q.,
thrombosis and occlusion in the post-implant period and
scarring of implant tissue. The development of
alternative, tissue-based heart valves for
transplantation is necessary due to unmet patient demands
to improve upon existing heart valve technologies, which
are mechanical valves requiring the constant use of
anticoagulants and glutaraldehyde fixed tissue valves
which eventually experience calcification.
Previous attempts at producing artificial tissues
and organs have met limited success. Orton, U.S. Patent
No. 5,192,312 describes treating a transplant tissue
sample with exogenous basic fibroblast growth factor
(BFGF) and repopulating the tissue with cells, preferably
allogeneic or autogenous fibroblasts, ostensibly to avoid
immunological rejection. The heart valves and heart
leaflets may be sterilized with lethally effective doses
of x-rays or with antibiotics, antibacterials and
cytotoxic agents. According to Orton, the addition of
bFGF to the tissue in vitro is critical in this system,
and is essential for causing the graft-populating cells
to migrate into the tissue and proliferate in response to
the growth factor, and populate the tissue. However,
Orton only demonstrates the system on small pieces of the
valve fixed in a petri dish and does not show production
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WO96/08213 ~ PCT~S95/11395
of a functional heart valve or any of the alleged
advantages if implanted in vivo, e.q., avoidance of
immunological rejection, and reduction in hardening
and/or scarring of the valve transplant. -
Livesey et al., U.S. Patent No. 5,336,616 relates toa method of producing a transplantable tissue graft for
processing and preserving acellular, collagen-based
tissue matrix for transplantation. The method described
involved processing biological tissues with a stabilizing
solution to prevent osmotic, hypoxic, autolytic and
proteolytic degradation and to control contamination.
The tissues were decellularized with EDTA, CHAPS or a
zwitterionic detergent, SDS or anionic/nonionic
detergent, followed by treatment with a cryoprotectant
such as DMSO, propylene glycol, butanediol, raffinose,
polyvinyl pyrrolidone, dextran or sucrose and vitrified
in liquid nitrogen. Thereafter, the tissues were
subjected to a dry stabilization procedure involving
molecular distillation drying under nitrogen gas,
followed by rehydration with buffered solution. Each of
the methods has limitations and therefore it is essential
that very stringent measures be taken to preserve the
biological properties of the material and avoid toxicity
resulting from reagents used during processing.
3. 8~MMARY OF T~E lNV ~:N ~-lON
The present invention relates to transplantable
cardiac tissue or bioprosthetic grafts composed of human
cells grown on three-dimensional frameworks, scaffolds or
matrices, a method of culturing human cells on such
frameworks and uses of such three-dimensional cell
cultures. In accordance with the invention, stromal
cells, including but not limited to human fibroblasts,
are inoculated and grown on a three-dimensional
frameworks, such as intact heart valves, aortic walls and
leaflets, or other biological scaffolding suitable for
reconstructing a valve or valve components, including for
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WO96/08213 PCT~S95/11395
example, but not limited to the pericardium or the small
intestinal submucosa or biodegradable frameworks or
matrices. The preferred three-dimensional framework may
be prepared from intact porcine heart valves, aortic wall
tissue, or leaflets which are decellularized (at -20C to
-70C or with detergents and enzymes) and sterilized by:
chemical methods including, but not limited to, ethylene
oxide and peracetic acid; irradiation including, but not
limited to, gamma and electron beam; and steam
sterilization including, but not limited to autoclaving.
No viable cells remain in the decellularized/sterilized
tissue samples which are used as a scaffold or framework
for culturing the stromal cells.
The stromal cells which are inoculated onto the
scaffold, may include dermal or cardiac fibroblasts,
and/or cells capable of producing collagen types I and
III, and in some instances, elastin, which are typically
produced in heart valves. (See Table I). The stromal
cells and connective tissue proteins naturally secreted
by the stromal cells attach to and substantially envelope
the three-dimensional framework or construct, having
interstitial spaces bridged by the stromal cells. The
living stromal tissue so formed provides the support,
growth factors, and regulatory factors necessary to
sustain long-term active proliferation of stromal cells
in culture and/or cultures implanted in vivo. When grown
in this three-dimensional system, the proliferating cells
mature and segregate properly to form components of adult
tissues analogous to counterparts in vivo.
In another embodiment of the invention, the stromal
cells can be genetically engineered to express a gene
product beneficial for successful and/or improved
transplantation. For example, the stromal cells can be
genetically engineered to express anticoagulation gene
products to reduce the risk of thromboembolism, or anti-
inflammatory gene products to reduce the risk of failure
due to inflammatory reactions. For example, the stromal
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WO96/08213 ~ PCT~S95/11395
cells can be genetically engineered to express tissue
plasminogen activator (TPA), streptokinase or urokinase
to reduce the risk of clotting. AlternatiVely, the
stromal cells can be engineered to express anti- -
inflammatory gene products, e.q., peptides or
polypeptides corresponding to the idiotype of
neutralizing antibodies for tumor necrosis factor (TNF),
interleukin-2 (IL-2), or other inflammatory cytokines.
Preferably, the cells are engineered to express such gene
products transiently and/or under inducible control
during the post-operative recovery period, or as a
chimeric fusion protein anchored to the stromal cell,
e.a., a chimeric molecule composed of an intracellular
and/or transmembrane domain of a receptor or receptor-
like molecule, fused to the gene product as the
extracellular domain.
In another alternative, the stromal cells can be
genetically engineered to "knock out" expression of
factors or surface antigens that promote clotting or
rejection. For example, expression of fibrinogen, von
Willebrands factor or any cell surface molecule that
binds to the platelet ~2B~-3 receptor can be knocked out
in the stromal cells to reduce the risk of clot
formation. Likewise, the expression of MHC class II
molecules can be knocked out in order to reduce the risk
of rejection of the graft.
In yet another embodiment of the invention, the
three-dimensional culture system of the invention may
afford a vehicle for introducing genes and gene products
in vivo to assist or improve the results of the
transplantation and/or for use in gene therapies. For
exa~ple, genes that prevent or ameliorate symptoms of
valvular disease such as thrombus formation, inflammatory
reactions, fibrosis and calcification, may be
underexpressed or overexpressed in disease conditions.
Thus, the level of gene activity in the patient may be
increased or decreased, respectively, by gene replacement
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WO96/08213 PCT~S95/11395
therapy by adjusting the level of the active gene product
in genetically engineered stromal cells.
In a specific embodiment exemplified by the examples
in Section 6, infra, human dermal fibroblasts were-grown
in the three-dimensional culture systems of the
invention. Porcine aortic walls and leaflets were chosen
because they are currently used in replacement therapy of
heart valves. Particular benefits were achieved in
porcine aortic wall and leaflet cultures where
proliferation of human fibroblasts occurred, and
production of tissue similar to human matrix proteins in
the aortic walls and leaflets was detected. These
characteristics were monitored by analyzing the
recellularized or remodeled constructs for cell
distribution (histological analyses), cell viability (MTT
assays), cell proliferation (3H-Thymidine labeling or BrdU
incorporation), protein production (3H-proline labeling,
35S-cysteine/methionine labeling), and protein
immunohistochemistry. The results from these studies
showed that human dermal fibroblasts were able to
colonize the porcine scaffolding of leaflets, aortic wall
biopsies and intact valves, cultured and grown over
several time intervals, for example, but not limited to
2, 4, 8 and 18 weeks. The present invention, thus,
relates to a method of repopulating porcine aortic walls
and leaflets with human fibroblasts to produce human
matrix proteins in which the porcine aortic leaflets and
walls are first sterilized with peracetic acid (or by
other chemical means such as ethylene oxide) or by
radiation with an electron beam (or by gamma irradiation)
or by steam (autoclaving).
In the examples described infra, human fibroblasts
were grown in culture on frameworks or constructs,
composed of porcine aortic valves, walls and leaflets
which had been decellularized and sterilized. When_
implanted in vivo, such frameworks or constructs allow
adequate nutrient and gas exchange to the cells until
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W096/08213 PCT~$9S/ll3s5
engraftment and vascularization at the site of
engraftment occurs. The advantage of adding human
fibroblasts to the three-dimensional, decellularized
porcine scaffolds or ~iodegradable constructs, is that
colonization of the porcine scaffolding results in a
valve implant with living cells which produce biological
factors that may stimulate host cells to endothelize the
implant and stimulate host cardiac fibroblasts to
integrate into the implant. The net result is
enhancement of host-graft take. Another advantage of
adding human fibroblasts is that cultures can be
maintained under sterile conditions without inhibiting
the growth of human fibroblasts, which grow in various
types of frameworks or constructs usually pretreated with
detergents or enzymes and sterilized with peracetic acid
or irradiation with the electron beam. ~urthermore,
heart valves colonized with functional human cells are
less likely to be su~ject to immunological rejection and
thus are superior to those heart valves which are covered
with xenogeneic cells prepared for use in replacement
therapy.
It is an object of the present invention to
construct a heart valve from human foreskin or cardiac
fibroblasts and porcine heart valve and/or aortic walls
and leaflets, which no longer contains porcine cells but
becomes a humanized porcine heart valve or a
recellularized heart valve suitable for transplantation
in humans. Such an approach provides an improved method
and means of designing, constructing and utilizing aortic
walls and leaflets, intact heart valves othèr biological
scaffolding suitable for reconstructing a valve or valve
component (e.c., pericardium, small intestinal submucosa,
etc.) and biodegradable frameworks, as scaffolding for
growth and implantation of human fibroblasts in ~itro.
It is further the object of the invention to
construct a heart ~alve consisting of human cells and
human tissue matrix proteins made by human dermal or
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WO96/08213 ~ ~ g ~ ~ ~ PCT~S95/11395
cardiac fibroblasts and a completely or nearly complete
bioresorbable/biocompatible polymer scaffolding in the
shape of different types of valves or their components,
for example, but not limited to aortic, pulmonary,-
mitral, and tricuspid valves. Such an approach provides
bioprosthetic or transplantable tissues, which can be
utilized for cell growth, both in vitro and in vivo, to
replace or reconstruct degenerated and dysfunctional
heart valves in human patients.
It is a still further object of this invention to
use human dermal or cardiac fibroblasts to colonize the
porcine aortic leaflets and wall biopsies or other
biological scaffolding suitable for reconstructing a
valve or valve components (e.a., pericardium, small
intestinal submucosa, etc.), and remain metabolically
viable with the result that all porcine cells native to
the leaflet and wall tissues or other biological
scaffolds, are either eliminated (decellularized) or
nonviable (dead). Such an approach provides an in vitro
system in which human fibroblast cells retain their
morphology and cell function for the secretion of
bioactive molecules normally produced in the body by the
cells of the aortic walls and leaflets or the intact
heart valve or the pulmonary, mitral, and tricuspid
valves.
The present invention relates to methods and
biological tissue prothesis or valves for the treatment
of valvular heart disease, including, but not limited to,
aortic stenosis, aortic regurgitation, mitral stenosis,
mitral regurgitation, pulmonary valve disease, tricuspid
valve disease, multivalvular disease, tricuspid valve
disease, Marfan syndrome and artificial valve disease.
4. BRIEF DE8CRIPTION OF THE DRAWINGR
Figure l is a photograph of autoradiographed proteins
synthesized by human dermal fibroblasts post seeding onto
porcine aortic leaflets and walls.
g
W O 96/08213 ~ PC~rnUS95111395
Figure 2 is a photograph of hematoxylin and eosin stained
tissue sections: a) a fresh porcine leaflet (the cardiac
fibroblast nuclei native to the tissue appear purple in
coloration); b) a detergent and/or enzyme extracted
porcine leaflet (no porcine cell nuclei are detected
after chemical treatment); c) a detergent and/or enzyme
extracted porcine leaflet cultured with human fibroblasts
for 18 weeks (the dermal human fibroblasts are present in
the porcine matrix). (Stained with Hematoxylin/Eosin.)
( lOx) .
Figure 3 is a photograph of a porcine leaflet seeded with
human dermal fibroblasts and cultured for 4 weeks.
(Sta~ned with Hematoxylin/Eosin.)
Figure 4 is a bar graph showing that in three sample sets
(#1-3) of detergent and/or enzyme extracted leaflets with
or without fibroblasts, only the leaflets which are grown
with fibroblasts incorporated 3H-thymidine, indicating
that the fibroblasts were proliferating.
Figure 5 is a SDS gel autoradiograph analysis showing
protein bands: non-viable porcine leaflet (lane 1) and
wall biopsy (lane 2) seeded with human fibroblasts show
protein synthesis, whereas unseeded, non-viable porcine
leaflet (lane 3) and porcine wall biopsy (lane 4) show no
activity. Fresh, viable porcine leaflet (lane 5) and
wall biopsy (lane 6) seeded with human fibroblasts have
similar patterns to fresh, viable, unseeded porcine
leaflet (lane 7) and wall biopsy (lane 8).
Figure 6. Porcine leaflet and wall (negative controls)
a,b, respectively) stained with serum only and showed no
background staining. Porcine leaflet stained with human
tenascin (c) and porcine wall stained with human
fibroblast antibody (d). Both c and d show no species
cross reactivity. Whole humanized porcine valve
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WO96/08213 ~ PCT~S95/11395
constructs cultured for 4 weeks under dynamic flow showed
positive staining for human tenascin in the leaflet,
wall, and muscle bar (e,g,i) and positive staining for
human fibroblasts in the leaflet, wall, and muscle bar
(f,h,j)-
Figure 7 is a photograph of autoradiographed proteinincorporation of human fibroblasts after dynamic culture
on porcine aortic leaflets.
Figure 8 is a photograph depicting human fibroblast
proliferation on a porcine matrix which was previously
decellularized by detergent and/or enzyme treatment. The
proliferating cells were labeled with Brdu and detected
using an antibody to Brdu and a visualization kit. The
labeled cells proliferating on the tissues were grown
under dynamic flow conditions. Brdu labeling occurred
during the last 72 hr of a 4 week culture period.
Figure 9 is a photograph depicting decellularized
(detergent and/or enzyme) + electron beamed valves seeded
with human fibroblasts under pulstile (left) and non-
pulstile (right) dynamic flow conditions. Both valves
were cultured dynamically for l week, then stained with
MTT. The valve seeded with pulstile flow conditions had
greater and more uniform fibroblast attachment.
5. DET~TT~Fn DE8CRIPTION OF ~HE lNv~l.,lON
The present invention relates to transplantable
cardiac tissue constructs or bioprosthetic grafts grown
in three-dimensional frameworks, a method of culturing
human cells on such frameworks and uses of such three-
dimensional, recellularized tissue constructs grown in
cultures. In accordance with the invention, stromal
cells, including but not limited to human fibroblasts,
are inoculated and grown on a three-dimensional framework
or construct of intact heart valves, aortic walls and
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WO96/08213 ~ 9 ~ PCT~S95/11395
leaflets or other biological scaffolding suitable for
reconstructing a valve or valve components, for example,
including but not limited to the pericardium or the small
intestinal submucosa or biodegradable frameworks. -Cells
grown on a three-dimensional framework, in accordance
with the present invention, grow to form a cellular
tissue-matrix which resembles tissue found in vivo to a
greater degree than previously described. The three-
dimensional cell culture system treated with human
stromal cells is applicable to the proliferation of
different types of cells and formation of a number of
different tissues, including ~ut not limited to aortic
walls and leaflets, or intact heart valves, pulmonary,
mitral, and tricuspid valves. In addition, the stromal
cells grown in the system may be genetically engineered -
to produce gene products beneficial to transplantation,
e.a., anti-coagulation factors, e.a., TPA, streptokinase,
etc., or anti-inflammatory factors, e.q., anti-TNF, anti-
IL-2, etc. Alternatively, the stromal cells may be
genetically engineered to "knock out" expression of
native gene products that promote platelet binding and -
clot formation, e.q., fibrinogen, von Willebrands factor,
or "knock out" expression of MHC in order to lower the
risk of rejection. In addition, the stromal cells may be
genetically engineered for use in gene therapy to adjust
the level of gene activity in a patient to assist or
improve the results of the transplantation.
The use of human foreskin fibroblasts in the three-
dimensional tissue constructs has a variety of advantages
and applications. For example, for a variety of cells
and tissues, such as porcine heart valves, aortic walls
and leaflets, the chordae tendinea in the mitral and
tricuspid valve, skin, ligaments, tendons, etc., the
three-dimensional tissue constructs can be produced at a
rapid rate and may itself ~e transplanted or implanted
into a living organism without undue delay. The three-
dimensional tissue constructs may also be used in vitro
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- IPEAIUS 26 NO~'96
for testing the effectiveness or cytotoxicity of
pharmaceutical agents, screening compounds for use in
treatment of clotting or thromboembolism, as
anticoagulants, as anti-inflammatory agents, as anti-
calcification agents or as endothelialization agents.
In yet another application, the three-dimensional tissue
construct system may be cellularized within a
"bioreactor" to produce a valve or valve component with
leaflet mobility and full valve function. For example,
an intact valve comprising of leaflets attached to the
wall, may be assembled as a three-dimensional framework,
inoculated with human stromal cells and maintained in
recirculating culture medium regulated by a peristaltic
or pneumatic pump which also keeps the leaflets or tissue
sheets/patches in a dynamic state. The bioreactor
provides a closed system free from problems of
contamination during procedures involving sterilization,
seeding, culturing, shipping and/or testing valve
function.
The methods for culturing cells including human
dermal fibroblasts on aortic walls and leaflet cells or
intact heart valves or other biological scaffolding
suitable for reconstructing a valve or valve components,
for example, but not limited to the pericardium or the
small intestinal submucosa or biodegradable frameworks,
as a three-dimensional biological or synthetic framework
or construct which can be used in accordance with the
invention are described in applicants' co-pending
application Serial No. 08/304,062 filed September 12,
1994; which is a continuation-in-part of Serial No.
08/254,096 filed June 6, 1994; which is a continuation-
in-part of Serial No. 08/131,361 filed October 4, 1993
(U.S. Patent No. 5,443,950), U.S. Patent No. 5,041,138 by
Vacanti et al., and application Serial No. 07/509,952
filed April 16, 19g0 by Vacanti et al., each of which is
incorporated by reference herein in its entirety.
AMENDED SHEET
WO96/08213 ~ PCT~S95/11395
Methods for the treatment of valvular heart disease,
including, but not limited to, aortic stenosis, aortic
regurgitation, mitral stenosis, mitral regurgitation,
pulmonary valve disease, tricuspid valve disease, ~
multivalvular disease, tricuspid valve disease, Marfan
syndrome and artificial valve disease, are described.
5.1. ESTABT.T~HM~NT OF THREE-DIMENSIONAL
FRAMEWORR
The three-dimensional framework for use in the
present invention may be of any material and/or shape
that: (a) allows cells to attach to it (or can be
modified to allow cells to attach to it); and (b) allows
cells to grow in more than one layer. It is preferred
that allogeneic and xenogeneic aortic walls and leaflets
or ir.tact heart valves or other biological scaffolding
suitable for reconstructing a valve or valve components,
for example, but not limited to the pericardium or the
small intestinal submucosa or biodegradable frameworks,
obtained from a variety of mammals, including but not
limited to, man, pig, cow, sheep or dog, may be used.
The porcine leaflets and aortic biopsies may be used in
the following forms: irradiated or chemically treated or
steam treated (sterilized); decellularized (for example,
detergent and/or enzyme treated), extracted and
sterilized; and valve tissue with nonviable cells and
other biological tissues, for example, but not limited
to, pericardium or small intestinal submucosa
(accomplished by such procedures as freezing at -20C to
-70C, or by repeated freezing and thawing).
The methods for decellularizing the aortic walls and
leaflets or intact valves or other biological scaffolding
suitable for reconstructing a valve or valve components,
which can be used in accordance with the invention,
include, but are not limited to the methods described in
U.S. Patent No. 5,336,616 and U.S. Patent No. 4,776,853,
which are incorporated herein by reference in their
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~ PCT~S9S/11395
W096/08213
entirety. ~or exampl~, the ti~ueQ can be decellularized
with EDTA, CHAPS or a zwitterionic detergent, f~llo~ed by
treatment with a cryoprotectant such as DMSo, propylene
glycol, butanediol, raf~inose, paly~inyl pyrrolidone,
dextran or sucro~e and vitri~ied in liquid ni~-~y~..
Alternatively, the ti~sue sample can be su~ject~d to
enzymatic dige~tion andJor extracting With reagents that
break down the cellular ~embranes and allow remoYal of
cell conten~ yA~rlec of aetergents include non-ionic
detergents ~or example, TRI~ON X-lOO, octylphenoxy
polyethoxyethanol, (Roh~ and Haas); BRIJ-35, a
polyethoxyethanol lauryl ether (Atlas Chemical Co.),
TWEEN 20, a polyethoxyethanol corbitan monolaureate tRohm
and HaaQ), LUBROL-PX, or polyethylene lauryl eth~r (Rohm
and Haas)~; and ionic detergents (for exa~ple, sodium
dodecyl sulphate, sulfated ~igher aliphatic alcohol,
sulfonated alkane and sulfona~ed alkylarene containing 7
to 22 carbon atoms in a branched or unbranched chain).
Th~ ~n2yme~ used may include nucleases (for example,
deoxyri~onucleasQ and r~bonuclease), protea~es,
phospholipa~es and lipase~. The tissues in the invention
can also be decellularized u~ing p~ysical procedure~ ~uch
as ultrasonic treatment or o~motic ~hock, or by chemical
treatment using peracetic aaid.
The three-d~mensional framew~rk may also be composed
of completely or nearly complete bio~e~orbablel.
bioco~patible polymer ~ffolaing in the shape of ~arious
different types of val~e~, $ncluding but not lim~ted ~o,
aortic, pulmonary, mitral, and tricuspid valves and valve
~Q~onents of each type. The bio~egradable scaffold6,
constructs, ~rameworks or mat~ice~ may be ~J ~ P~ of
materials such a~ polyglyco~ic acid, catgut ~U~ULe
material, hyalur~nic acid, cellulo6e, collagen (ln the
form of ~ponges, braid~, or woven threads, etc.),
gelat~n, or other na~urally o~ L ing biodegradable
material~ or synt~etic material~, including for example,
a variety oS polyhydroxyalkanoates. Such framework or
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WO96/08213 ~ PCT~S95/11395
constructs may be molded into the shape of heart valves
or repair sheets/patches prior to inoculation of human
cells. Where possible, however, it is most preferable to
use a three-dimensional construct of the tissue of
origin, for example, the aortic walls and leaflets or
intact heart valves.
The invention is based in part, on the discovery
that the three-dimensional system supports the
proliferation, migration, differentiation, and
segregation of cells in culture in vitro to form
components of tissues analogous to counterparts found in
vivo. The human cells added to the scaffolds repopulate
the porcine valve without the need for exogenously added
growth factors. This is contrary to Orton's teachings
which show that leaflet tissue not treated with bFGF
remained acellular. The use of growth factors (for
example, but not limited to, aFGF, ~FGF, insulin growth
factor or TGF-betas), or natural or modified blood
products or other bioactive biological molecules (for
example, but not limited to, hyaluronic acid or
hormones), even though not absolutely necessary in the
present invention, may be used to further enhance the
reconstitution of the porcine or other biological
scaffolding.
Although the applicants are under no duty or
obligation to explain the mechanism by which the
invention works, a number of factors inherent in the
three-dimensional culture system may contribute to its
success:
(a) The three-dimensional framework provides a
greater surface area for protein attachment, and
consequently, for the adherence of stromal cells.
(b) Because of the three-dimensionality of the
framework, stromal cells continue to actively grow, in
contrast to many cells in monolayer cultures, which grow
to confluence, exhibit contact inhibition, and cease to
grow and divide. The elaboration of extracellular matrix
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WO96/08213 ~ PCT~S95/11395
proteins and secretion of growth and regulatory factors
by replicating stromal cells may be partially responsible
for stimulating proliferation, maintaining normal tissue
-differentiation and regulating differentiation of-cells
in culture.
(c) The three-dimensional framework allows for a
spatial distribution of cellular elements which is more
analogous to that found in the counterpart tissue in
ViVO .
(d) The increase in potential volume for cell
growth in the three-dimensional system may allow the
establishment of localized microenvironments conducive to
cellular maturation.
(e) The three-dimensional framework maximizes cell-
cell interactions by allowing greater potential for
movement of migratory cells.
(f) It has been recognized that maintenance of a
differentiated cellular phenotype requires not only
growth/differentiation factors but also the appropriate
cellular interactions. The present invention effectively
recreates the tissue microenvironment.
The three-dimensional stromal support, the culture
system itself, and its maintenance, as well as various
uses of the three-dimensional cultures are described in
greater detail in the subsections below.
5.2. E8T~RTT8~M~NT OF THREE-DIMEN8IONAL
8TROMAL TISS~E
Stromal cells comprising fibroblasts, with or
without other stromal cells and elements described below,
are inoculated onto the three-dimensional framework.
Human fibroblasts may be added to the culture prior to,
during or subsequent to inoculation of other stromal
cells. The concentration of fibroblasts maintained in
the cultures can be monitored and adjusted appropriately
to optimize growth and to regulate scaffold colonization.
Alternatively, stromal cells that are genetically
WO96108213 ~ PCT~S95/1139S
engineered to express and produce factors similar to
those produced by cells of the heart valve, may be
included in the inoculum. These cells could serve as a
source of protein factor(s) in the culture. Preferably,
the gene or coding sequence for factor(s) would be placed
under the control of a regulated promoter, so that
production of factor(s) in culture can be controlled.
The genetically engineered cells will be screened to
select those cell types: l) that bring about
amelioration of blood clotting, coagulation,
thromboembolism and inflammatory reactions in vivo, and
2) escape immunological surveillance and rejection.
Stromal tissue comprising dermal fibroblasts,
cardiac fibroblasts and cells capable of producing
collagen type I and III, elastin and other heart valve
matrix proteins, for example, but not limited to
fibronectin and glycosaminoglycans, are used to grow in
vitro, transplantable tissue or bioprosthetic heart
valves. Stromal cells such as fibroblasts can be
obtained in quantity rather conveniently from skin, human
foreskin, heart tissue or any appropriate organ. Fetal
and neonatal fibroblasts can be used to form a "generic"
three-dimensional stromal tissue construct that will
support the growth of a variety of different cells and/or
tissues. Fibroblasts may be readily isolated by
disaggregating an appropriate organ or tissue which is to
serve as the source of the fibroblasts. This may be
readily accomplished using techniques known to those
skilled in the art. For example, the tissue or organ can
be disaggregated mechanically and/or treated with
digestive enzymes and/or chelating agents that weaken the
connections between neighboring cells making it possible
to disperse the tissue into a suspension of individual
cells without appreciable cell breakage. Enzymatic
dissociation can be accomplished by mincing the tissue
and treating the minced tissue with any of a number of
digestive enzymes either alone or in combination. These
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WO96/08213 PCT~S9Sl11395
include but are not limited to trypsin, chymotrypsin,
collagenase, elastase, hyaluronidase, pronase, etc.
Mechanical disruption can also be accomplished by a
number of methods including, but not limited to the use
of grinders, blenders, sieves, homogenizers, or pressure
cells to name but a few. For a review of tissue
disaggregation techniaues, see Freshney, Culture of
Animal Cells. A Manual of Basic Technique, 2d Ed., A.R.
Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.
Once the tissue has been reduced to a suspension of
individual cells, the suspension can be fractionated into
subpopulations from which the fibroblasts and/or other
stromal cells and/or elements can be obtained. This also
may be accomplished using standard techniques for cell
separation including but not limited to cloning and
selection of specific cell types, selective destruction
of unwanted cells (negative selection), separation based
upon differential cell agglutinability in the mixed
population, freeze-thaw procedures, differential
adherence properties of the cells in the mixed
population, filtration, conventional and zonal
centrifugation, centrifugal elutriation (counter-
streaming centrifugation), unit gravity separation,
counter current distribution, electrophoresis and
fluorescence-activated cell sorting. For a review of
clonal selection and cell separation techniques, see
Freshney, Culture of Animal Cells. A Manual of Basic
Techniaues, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch.
11 and 12, pp. 137-168.
The isolation of fibroblasts may, for example, be
carried out as follows: fresh tissue samples are
thoroughly washed and minced in Hanks balanced salt
solution (HBSS) in order to remove serum. The minced
tissue is incubated from 1-12 hours in a freshly prepared
solution of a dissociating enzyme such as trypsin. After
such incubation, the dissociated cells are suspended,
pelleted by centrifugation and plated onto culture
-- 19 --
WO96/08213 ~ PCT~S95/11395
dishes. Fibroblasts will attach before other cells,
therefore, appropriate stromal cells can be selectively
isolated and grown. The isolated fibroblasts can then be
grown to confluency, lifted from the confluent culture
and inoculated onto the three-dimensional support (see,
Naughton et al., 1987, J. Med. 18(3&4):219-250).
Inoculation of the three-dimensional matrix with a high
concéntration of stromal cells, e.a., approximately lO6 to
5 x 107 cells/ml, will result in the establishment of the
three-dimensional stromal construct in shorter periods of
time.
Again, where the cultured cells are to be used for
transplantation or implantation in vivo it is preferable
to obtain the stromal cells from the patient's own
tissues. However, it is also possible to use allogeneic
compatible human cells, without significant rejection
reactions following transplantation. The growth of cells
in the presence of the three-dimensional stromal support
matrix may be further enhanced by adding to the matrix,
or coating the matrix support with specific amino acids,
proteins, glycoproteins, glycosaminoglycans, a cellular
matrix, andtor other materials.
After inoculation of the stromal cells, the three-
dimensional matrix should be incubated in an appropriate
nutrient medium. Many commercially available media such
as DMEM, RPMI 1640, Fisher's Iscove's, McCoy's, and the
like may be suitable for use. It is preferable that the
three-dimensional stromal matrix be suspended or floated
in the medium during the incubation period in order to
maximize proliferative activity. The container in this
protocol is kept stable in the incubator, i.e., under
static conditions (no circulating or flowing fluid). In
addition, the culture should be "fed" periodically to
remove the spent media, depopulate released cells, and
add fresh nutrients. The concentration of fibroblasts
may be adjusted during these steps. These procedures are
greatly facilitated when carried out using a bioreactor,
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WO96/08213 p~ sslll395
which is a closed system housing the three-dimensional
framework inoculated with stromal cells. A bioreactor
reduces the possibility of contamination, maintains the
cultures in recirculating, continuous culture medium and
keeps the leaflets in a dynamic state by opening and
closing them. Nore particularly, the U.S. patent
application entitled "Apparatus and Method for
Sterilizing, Seeding, Culturing, Storing, Shipping, and
Testing Tissue, Synthetic or Mechanical Heart Valves or
Valve Segments" and filed concurrently herewith by the
assignee of the present application, teaches the mode of
operation of the bioreactor and, is incorporated by
reference herein.
During the incubation period, the stromal cells will
attach and proliferate along the three-dimensional
framework before beginning to migrate into the depths of
the matrix. One objective is to grow the cells to an
appropriate degree which reflects the amount of stromal
cells present in the in vivo tissue. A second objective
is to regulate the number of cells in the inoculum and/or
their growth on the scaffold such that the amount of
scaffold colonization can be controlled as desired, and
reproducibly.
The openings of the non-tissue framework or
constructs should be of an appropriate size to allow the
stromal cells to stretch across the openings.
Maintaining actively growing stromal cells which stretch
across the framework enhances the production of growth
factors which are elaborated by the stromal cells, and
hence will support long term cultures. For example, if
the openings are too small, the stromal cells may rapidly
achieve confluence but be unable to easily exit from the
mesh; trapped cells may exhibit contact inhibition and
cease production of the appropriate factors necessary to
support proliferation and maintain long term cultures.
If the openings are too large, the stromal cells may be
unable to stretch across the opening; this will also
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W O 96108213 ~ PC~rnUS95/11395
decrease stromal cell production of the appropriate
factors necessary to support proliferation and maintain
long term cultures. When using a mesh type of matrix, as
exemplified herein, we have found that openings ranging
from about 150 ~m to about 220 ~m will work satisfactory.
However, depending upon the three-dimensional structure
and intricacy of the framework, other sizes may work
equally well. In fact, any shape or structure that
allows the stromal cells to stretch and continue to
replicate and grow for lengthy time periods will work in
accordance with the invention.
The human dermal fibroblasts exhibit a varied
affinity for the different types of porcine tissue
matrices. The greatest fibroblast colonization occurs
when using a porcine matrix that is detergent and/or
enzyme extracted. Additionally, the amount of fibroblast
colonization in the porcine tissue correlates with time.
Different proportions of the various types of
collagen deposited on the framework can be manipulated.
For example, for optimal growth of transplantable or
bioprosthetic heart valves, collagen types I and III are
preferably deposited in the initial matrix. The
proportions of collagen types deposited can be
manipulated or enhanced by selecting fibroblasts or cells
which elaborate the appropriate collagen type. This can
be accomplished using monoclonal antibodies of
appropriate isotypes or subclass that are capable of
activating complement, and which define particular
collagen type. These antibodies and complement can be
used to negatively select the fibroblasts which express
the desired collagen type. Alternatively, the stroma
used to inoculate the matrix can be a mixture of cells
which synthesize the appropriate collagen types desired.
The distribution and origins of the five types of
collagen is shown in Table I. Thus, for the growth and
preparation of heart valves, fibroblasts are the
preferred cells for the present invention.
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PCT~S95/11395
WO96/08213
TABLE I
DISTRIBUTIONS AND ORIGINS OF
THE FIVE TYPES OF COLLAGEN
Tissue Distribution Cells of Oriqin
I connective tissue; reticular cells;
collagen fibers smooth muscle
cells
Fibrocartilage
Bone Osteoblast
Heart Valve Fibroblasts
Dentin Odontoblasts
II Hyaline and elastic Chondrocytes
cartilage
Yitreous body of eye Retinal cells
III Loose connective tissue; Fibroblasts and
reticular fibers reticular cells
Papillary layer of
dermis
Blood vessels . Smooth muscle
cells;
endothelial
cells
IV Basement membranes Epithelial and
endothelial
cells
Lens capsule of eye Lens fibers
V Fetal membranes; Fibroblast
placenta
Basement membranes
WO96/08213 ~ ~ Q ~ PCT~S95/11395
Bone
Smooth muscle Smooth muscle
cells
During incubation of the three-dimensional stromal
construct, proliferating cells may be released from the
framework. These released cells may stick to the walls
of the culture vessel where they may continue to
proliferate and form a confluent monolayer. This should
be prevented or minimized, for example, by removal of the
released cells during feeding, by coating the culture
vessel with substances such as silicone to decrease
cellular attachment, or by transferring the three-
dimensional stromal framework to a new culture vessel.
The presence of a confluent monolayer in the vessel will
"shut down" the growth of cells in the three-dimensional
framework and/or culture. Removal of the confluent
monolayer or transfer of the framework to fresh media in
a new vessel will restore proliferative act~ivity of the
three-dimensional culture system. Such removal or
transfers should be done in any culture vessel which has
a stromal monolayer exceeding 25~ confluency.
Alternatively, the culture system could be agitated
to prevent the released cells from adhering, or instead
of periodically feeding the cùltures, the culture system
could be set up so that fresh media continuously flows
through the system. The flow rate could be adjusted to
both maximize proliferation within the three-dimensional
culture, and to wash out and remove cells released from
the matrix, so that they will not adhere to the walls of
the vessel and grow to confluence.
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WO96/08213 PCT~S9~111395
5.3. USES OF THE TRANSPLANTABLE HUMAN
CEL~-COLONIZED ~EART VA~VES AND
8HEET8 GROWN IN THREE-DIMEN8IONAL
CULTURE 8YSTEM
The three-dimensional culture system of the
invention can be used in a variety of applications.
These include but are not limited to transplantation or
implantation of either the cultured tissue obtained from
the framework, or the cultured matrix itself in vivo;
screening the effectiveness and cytotoxicity of
pharmaceutical agents, blood related natural and modified
compounds, growth/regulatory factors, etc., in vitro;
elucidating the mechanism of certain diseases; studying
the mechanism by which drugs and/or growth factors
operate; gene therapy; and the production of biologically
actlve products, to name but a few.
5.3.1. TRANSPLANTATION IN VIVO
The biological heart valves produced in the three-
dimensional culture system of the invention can be used
in the treatment of aortic stenosis, aortic
regurgitation, mitral stenosis, mitral regurgitation,
pulmonary valve disease, tricuspid valve disease,
multivalvular disease, tricuspid valve disease, Marfan
syndrome and artificial valve disease.
Aortic stenosis is the obstruction to flow across
the aortic valve during left ventricular systolic
ejection. It can be caused by a congenital unicuspid or
bicuspid valve, rheumatic fever, or degenerative
calcification of the valve in the elderly. The incidence
of bicuspid aortic valve has been estimated at 4 in 1,000
live births, with males dominating over females at 4:1.
Campbell, M., and Kauntze, R., 1953, Br. Heart J. 15:179.
Leaflets often thicken by age 40 and almost invariably by
age 50, but calcium deposits are rarely detected before
40 years of age. Although symptoms generally occur late
in the course of aortic stenosis, 3 to 5 percent of
patients may die suddenly during an otherwise a
- 25 -
WO96/08213 ~ 8 ~ ~ PCT~S95/11395
symptomatic period. Thus, patients with any sign of
congestive heart failure, angina, or exertional syncope
in the presence of significant aortic valvular stenosis
should undergo aortic valve replacement promptly. In
addition, asymptomatic patients with significant aortic
valvular stenosis should be advised to have valve
replacement therapy.
Aortic regurgitation is the diastolic flow of blood
from the aorta into the left ventricle. It is caused by
incompetent closure of the aortic valve which results
from intrinsic disease of the cusp or from diseases
affecting the aorta. Acquired intrinsic diseases of the
aortic valve are either rheumatic or from bacterial
origin. In the Marfan syndrome the primary basis for
aortic insufficiency usually resides in the aorta, but
there may be prolapse of the aortic cusps due to
myxomatous changes. Infrequent changes are seen with
rheumatoid arthritis, systemic lupus erythematosus, and
trauma. Oh, W.M.C., Taylor, T.R. and Olsen, E.G.J.,
1974, Br. Heart J.-36:413. Patients with chronic aortic
regurgitation who are symptomatic are advised to have
surgery. The type of operation used depends primarily on
the etiology. In patients with diseases limited to the
valve, the operation is essentially as described above,
for aortic stenosis.
Mitral stenosis designates resistance to flow
through the mitral apparatus during diastolic filling of
the left ventricle. Resistance to diastolic flow across
the mitral valve can result from rheumatic valvulitis,
congenital stenosis, thrombus formation, atrial myxoma,
bacterial vegetations, and calcification in the valve, as
well as in the annulus. The decision to intervene
surgically in patients with mitral stenosis is based on
the anticipated necessity of valve replacement versus
valve reconstruction therapy.
Mitral regurgitation occurs when contraction of the
left ventricle ejects blood into left atrium as a result
- 26 -
WO96/08213 PCT~S95/11395
of abnormalities in the mitral valve apparatus. Acute
mitral regurgitation can be created from mechanical
disruption of the chordae tendineae, rupture of the
papillary muscle, or perforation of the leaflet.
Rheumatic fever, mitral valve prolapse and coronary
artery disease, such as left ventricular dilation,
calcified mitral annulus, heritable disorders (Marfan
syndrome, Ehlers-Danlos, osteogenesis), congenital heart
disease, systemic lupus erythematosus, rupture of
papillary muscle and perforation of leaflet, are the
predominant mechanisms for the incompetence of the mitral
valve. Replacement of the mitral valve, valve components
and/or other affected parts such as the chordae, is
required in cases of rheumatic involvement leading to
severe mitral regurgitation, mitral stenosis with loss of
pliability of the leaflets, and various other causes of
mitral regurgitation, such as infective endocarditis, and
in some cases in chronic heart disease. Calcification
and immobility of the leaflets are also indications for
valve replacement.
Pulmonary stenosis is created by obstruction to
systolic flow across the valve and is most commonly
congenital. It generally leads to pulmonary
regurgitation. Pulmonary valve replacement may be
performed for acquired conditions such as carcinoid heart
disease and infective endocarditis.
Tricuspid regurgitation develops when the tricuspid
valve allows blood to enter the right atrium during right
ventricular contraction. Tricuspid stenosis represents
obstruction to diastolic flow across the valve during
diastolic filling of the right ventricle. The main cause
of tricuspid and mitral regurgitation is the rupture of
one or more of the elements of the tensor apparatus, with
disruption of the papillary muscle and rupture of the
chordae tendineae. Replacement is necessary if the
changes in the leaflets and subvalvular mechanism are
- 27 -
WO96/08213 ~ PCTNS95111395
advanced, or if severe regurgitation cannot be relieved
by annulopolasty.
Multivalvular disease indicates obstruction and/or
incompetence of the aortic, mitral, and tricuspid -valves.
Rheumatic fever, connective tissue diseases, Marfan
syndrome, calcification of the mitral valve in the aging
patient and bacterial endocarditis remain important
causes in combined disease of the mitral and aortic
valves. In patients with severe and progressive symptoms
having evidence of disease at both the mitral and aortic
valves, both valves are generally repressed by surgery.
Artificial valve disease includes any abnormality of
a surgically implanted device to replace a diseased
cardiac valve. Artificial valve disease can result from
prosthetic dysfunction, thrombus formation, infection,
fibrosis, or calcification. Roberts, W.C., 1973, Prog.
Cardiovasc. Dis. l5:539. Congestive heart failure due to
mechanical valve dysfunction is the major indication for
replacement of a mechanical artificial valve.
Replacement of the prosthesis is indicated if the
symptoms cannot be controlled medically or if there is
evidence of progressive ventricular dysfunction.
The second most common operation performed in adults
is replacement of the aortic or mitral valve. The valves
produced in accordance with the invention may be
transplanted using similar, if not the same surgical
techniques, well known to those skilled in the art. The
procedure for the replacement of the aortic valve is
performed through a median sternum-splitting incision.
After cardiopulmonary bypass is begun, a vascular clamp
is placed across the distal ascending aorta. A sump
suction cannula is placed in the left atrium through an
incision in the right superior pulmonary vein to
decompress the left heart. A transverse incision is made
in the proximal aorta and the diseased valve is excised.
Horizontal mattress sutures are placed at the three
commissures for traction. Simple radial sutures are then
- 28 -
~ ~ ~ PCT~S95/11395
WO96/~213
placed along the annulus between traction sutures and
passed sequentially through the sewing ring of the valve
as they are inserted. When all sutures have been passed
through the sewing ring, the valve is lowered into
position and the sutures are tied and cut. The aortotomy
is closed with continuous sutures.
Coronary artery perfusion usually is not necessary
for single-valve replacement, provided it can be
accomplished in 60 minutes or less. Adequate myocardial
protection can be afforded by systemic hypothermia at
30 C, injection of cardioplegic solution into the
ascending aorta, and lavage of the heart by iced isotonic
solution before it is opened.
In case of a mitral valve replacement, tricuspid
valve replacement or a pulmonary valve replacement, the
procedure is modified accordingly and involves the same
technical maneuvers as outlined above. A detailed
description of the operative surgery used is described in
P.F. Nora, ed., OPerative SurgerY PrinciPles and
Techniques, 2nd ed. (1980) 326-327; J.W. Kirklin and B.G.
Barratt-Boyes, eds., Cardiac SurqerY MorPholoqy
Diaqnostic Criteria Natural HistorY Techniques
Results, and Indications, 2d ed. (1993) 498-507; and J.W.
Hurst and R.C. Schlant, eds., The Heart, Arteries and
Veins, 7th ed. (1990) 795-876.
For transplantation or implantation in vivo, either
portions of the culture or the entire three-dimensional
culture could be implanted, depending upon the type of
tissue involved. For example, three-dimensional heart
valve cultures can be maintained in vitro for long
periods. Section of tissues or the entire three-
dimensional tissue structure can be transplanted in vivo
in patients needing new heart valves.
Three-dimensional tissue culture implants may,
according to the inventions, be used to replace or
augment existing tissue, to introduce new or altered
- 29 -
WO96/08213 ~ ~3 ~ PCT~S95/11395
tissue, to modify artificial prostheses, or to join
together biological tissues or structures.
5.3.2. 8CREENING EFFE~lv~N~ AND
~ OXICITY OF COMPOUNDS IN VITRO
The three-dimensional cultures may be used in vitro
to screen a wide variety of compounds, for effectiveness
and cytotoxicity of pharmaceutical agents,
growth/regulatory factors, natural and modified blood
products, anticoagulants, clotting agents or anti-
calcification agents, etc. To this end, the cultures are
maintained in vitro and exposed to the compound to be
tested. The activity of a cytotoxic compound can be
measured by its ability to damage or kill cells in
culture. This may readily be assessed by vital staining
techniques. The effect of growth/regulatory factors may
be assessed by analyzing the cellular content of the
matrix, e.g., by total cell counts, and differential cell
counts. This may be accomplished using standard
cytological and/or histological techniques including the
use of immunocytochemical techniques employing antibodies
that define type-specific cellular antigens. The effect
of various drugs on normal cells cultured in the three-
dimensional system may be assessed.
5.3.3. GENE THERAPY
The three-dimensional culture system of the
invention may afford a vehicle for introducing genes and
gene products in vivo to assist or improve the results of
the transplantation and/or for use in gene therapies.
For example, the stromal cells can be genetically
engineered to express anticoagulation gene products to
reduce the risk of thromboembolism, or anti-inflammatory
gene products to reduce the risk of failure due to
inflammatory reactions. In this regard, the stromal
cells can be genetically engineered to express TPA,
streptokinase or urokinase to reduce the risk of
- 30 -
8 ~ ~
WO96/08213 PCT~S95/11395
clotting. Alternatively, the stromal cells can be
engineered to express anti-inflammatory gene products,
for example, peptides or polypeptides corresponding to
the idiotype of neutralizing antibodies for TNF, I-L-2, or
other inflammatory cytokines. Preferably, the cells are
engineered to express such gene products transiently
and/or under inducible control during the post-operative
recovery period, or as a chimeric fusion protein anchored
to the stromal cells, for example, a chimeric molecule
composed of an intracellular and/or transmembrane domain
of a receptor or receptor-like molecule, fused to the
gene product as the extracellular domain. In another
embodiment, the stromal cells could be genetically
engineered to express a gene for which a patient is
deficient, or which would exert a therapeutic effect,
e.q., HDL, apolipoprotein E, etc. The genes of interest
engineered into the stromal cells need to be related to
heart disease. For example, the stromal cells can be
engineered to express gene products that are carried by
the blood; e.q., cerebredase, adenosine deaminase, ~
antitrypsin. In a particular embodiment, a genetically
engineered valve culture implanted to replace the
pulmonary valve can be used to deliver gene products such
as ~-l-antitrypsin to the lungs; in such an approach,
constitutive expression of the gene product is preferred.
The stromal cells can be engineered using a
recombinant DNA construct containing the gene used to
transform or transfect a host cell which is cloned and
then clonally expanded in the three-dimensional culture
system. The three-dimensional culture which expresses
the active gene product, could be implanted into an
individual who is deficient for that product. For
example, genes that prevent or ameliorate symptoms of
various types of valvular heart diseases may be
underexpressed or down regulated under disease
conditions. Specifically, expression of genes involved
in preventing the following pathological conditions may
- 31 -
WO96108213 ~ 8 ~ ~ - PCr/US95111395
be down-regulated, for example: thrombus formation,
inflammatory reactions, and fibrosis and calcification of
the valves. Alternatively, the activity of gene products
may be diminished, leading to the manifestations of some
or all of the above pathological conditions and eventual
development of symptoms of valvular disease. Thus, the
level of gene activity may be increased by either
increasing the level of gene product present or by
increasing the level of the active gene product which is
present in the three-dimensional culture system. The
three-dimensional culture which expresses the active
target gene product can then be implanted into the
valvular disease patient who is deficient for that
product. "Target gene," as used herein, refers to a gene
involved in valvular disease in a manner by which
modulation of the level of target gene expression or of
target gene product activity may act to ameliorate
symptoms of valvular disease.
Further, patients may be treated by gene replacement
therapy during the post-recovery period after
transplantation. Heart valve constructs or sheets may be
designed specifically to meet the requirements of an
individual patient, for example, the stromal cells may be
genetically engineered to regulate one or more genes; or
the regulation of gene expression may be transient or
long-term; or the gene activity may be non-inducible or
inducible. For example, one or more copies of a normal
target gene, or a portion of the gene that directs the
production of a normal target gene protein product with
target gene function, may be inserted into human cells
that populate the three-dimensional constructs using
either non-inducible vectors including, but are not
limited to, adenovirus, adeno-associated virus, and
retrovirus vectors, or inducible promoters, including
metallothionein, or heat shock protein, in addition to
other particles that introduce DNA into cells, such as
liposomes or direct DNA injection or in gold particles.
-- 32 --
WO96/08213 - PCT~S95/11395
For example, the gene encoding the human complement
regulatory protein, which prevents rejection of the graft
by the host, may be inserted into human fibroblasts.
Nature 375: 89 (May, 1995).
The three-dimensional cultures containing such
genetically engineered stromal cells, e.a., either
mixtures of stromal cells each expressing a different
desired gene product, or a stromal cell engineered to
express several specific genes are then implanted into
the patient to allow for the amelioration of the symptoms
of valvular disease. The gene expression may be under
the control of a non-inducible (i.e., constitutive) or
inducible promoter. The level of gene expression and the
type of gene regulated can be controlled depending upon
the treatment modality being followed for an individual
patient.
The use of the three-dimensional culture in gene
therapy has a number of advantages. Firstly, since the
culture comprises eukaryotic cells, the gene product will
be properly expressed and processed in culture to form an
active product. Secondly, gene therapy techniques are
useful only if the number of transfected cells can be
substantially enhanced to be of clinical value,
relevance, and utility; the three-dimensional cultures of
the invention allow for expansion of the number of
transfected cells and amplification (via cell division)
of transfected cells.
A variety of methods may be used to obtain the
constitutive or transient expression of gene products
engineered into the stromal cells. For example, the
transkaryotic implantation technique described by Seldon,
R.F., et al., 1987, Science 236:714-718 can be used.
"Transkaryotic", as used herein, suggests that the nuclei
of the implanted cells have been altered by the addition
of DNA sequences by stable or transient transfection.
The cells can be engineered using any of the variety of
vectors including, but not limited to, intergating viral
- 33 -
W096/08213 v ~ PCT~S95111395
vectors, e.q., retrovirus vector or adeno-associated
viral vectors, or non-integrating replicating vectors,
e.a., papilloma virus vectors, SV40 vectors, adenoviral
vectors; or replication-defective viral vectors. -Where
transient expression is desired, non-integrating vectors
and replication defective vectors may be preferred, since
either inducible or constitutive promoters can be used in
these systems to control expression of the gene of
interest. Alternatively, integrating vectors can be used
to obtain transient expression, provided the gene of
interest is controlled by an inducible promoter.
Preferably, the expression control elements used
should allow for the regulated expression of the gene so
that the product is synthesized only when needed in vivo.
The promoter chosen would depend, in part upon the type
of tissue and cells cultured. Cells and tissues which
are capable of secreting proteins (e.q., those
characterized by abundant rough endoplasmic reticulum,
and golgi complex) are preferable. Hosts cells can be
transformed with DNA controlled by appropriate expression
control elements (e.q., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.)
and a selectable marker. Following the introduction of
the foreign DNA, engineered cells may be allowed to grow
in an enriched media, and then are switched to a
selective media. The selectable marker in the
recom~inant plasmid confers resistance to the selection
and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which, in turn,
can be cloned and expanded into cell lines. This method
can advantageously be used to engineer cell lines which
express the gene protein product.
Any promoter may be used to drive the expression of
the inserted gene. For example, viral promoters include
but are not limited to the CMV promoter/enhancer, SV 40,
papillomavirus, Epstein-Barr virus, elastin gene promoter
and ~-globin. If transient expression is desired, such
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~6~ Q
WO96/08213 PCT~S95/11395
constitutive promoters are preferably used in a non-
integrating and/or replication-defective vector.
Alternatively, inducible promoters could be used to drive
the expression of the inserted gene when necessary. For
example, inducible promoters include, but are not limited
to, metallothionein and heat shock protein.
Examples of transcriptional control regions that
exhibit tissue specificity for connective tissues which
have been described and could be used, include but are
not limited to: elastin or elastase I gene control
region which is active in pancreatic acinar cells (Swit
et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold
Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,
1987, Hepatology 7:425-515). The deposition of elastin
is correlated with specific physiological and
developmental events in different tissues, including the
heart valves. For example, atrioventricular valve cusps
are initially thick and fleshy in an embryo, and later in
the development are transformed into thin and fibrous
cusps. In developing arteries, elastin deposition
appears to be coordinated with changes in arterial
pressure and mechanical activity. Animals that contain
valves and ligamental structures that are elastic contain
elastin. The transduction mechanisms that link
mechanical activity to elastin expression involve cell-
surface receptors. Once elastin-synthesizing cells are
attached to elastin through cell-surface receptors, the
synthesis of additional elastin and other matrix proteins
may be influenced by exposure to stress or mechanical
forces in the tissue (for example, the constant movement
of the construct in the bioreactor) or other factors that
influence cellular shape.
Once genetically engineered cells are implanted into
an individual, the presence of TPA, streptokinase or
urokinase activity can bring about amelioration of
platelet aggregation, blood coagulation or
thromboembolism. This activity is maintained for a
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WO96/08213 ~ ~ 9 ~ 8 ~ ~ PCT~S95/11395
limited time only, for example, to prevent potential
complications that generally develop during the early
phase after valve implantation, such as, platelet
aggregation, blood clotting, coagulation or
thromboembolism. Alternatively, once genetically
engineered cells are implanted into an individual, the
presence of the anti-inflammatory gene products, for
example, peptides or polypeptides corresponding to the
idiotype of neutralizing antibodies for TNF, IL-2, or
other inflammatory cytokines, can bring about
amelioration of the inflammatory reactions associated
with valvular disease.
The stromal cells used in the three-dimensional
culture system of the invention may be genetically
engineered to "knock out" expression of factors or
surface antigens that promote clotting or rejection at
the implant site. Negative modulatory techniques for the
reduction of target gene expression levels or target gene
product activity levels are discussed below. "Negative
modulation", as used herein, refers to a reduction in the
level and/or activity of target gene product relative to
the level and/or activity of the target gene product in
the absence of the modulatory treatment. The expression
of a gene native to stromal cell can be reduced or
knocked out using a number of techniques, for example,
expression may be inhibited by inactivating the gene
completely (commonly termed "knockout") using the
homologous recombination technique. Usually, an exon
encoding an important region of the protein (or an exon
5' to that region) is interrupted by a positive
selectable marker (for example neo), preventing the
production of normal mRNA from the target gene and
resulting in inactivation of the gene. A gene may also
be inactivated by creating a deletion in part of a gene,
or by deleting the entire gene. By using a construct
with two regions of homology to the target gene that are
far apart in the genome, the sequences intervening the
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WO96/08213 PCT~S95111395
two regions can be deleted. Mombaerts, P., et al., l99l,
Proc. Nat. Acad. Sci. U.S.A. 88:3084-3087.
Antisense and ribozyme molecules which inhibit
expression of the target gene can also be used in-
accordance with the invention to reduce the level of
target gene activity. For example, antisense RNA
molecules which inhibit the expression of major
histocompatibility gene complexes (HLA) shown to be most
versatile with respect to immune responses. Still
further, triple helix molecules can be utilized in
reducing the level of target gene activity. These
techniques are described in detail by L.G. Davis, et al.,
eds, Basic Methods in Molecular BioloqY, 2nd ed.,
Appleton & Lange, Norwalk, Conn. 1994.
Using any of the foregoing techniques, the
expression of fibrinogen, von Willebrands factor, factor
V or any cell surface molecule that binds to the platelet
~2B~-3 receptor can be knocked out in the stromal cells
to reduce the risk of clot formation at the valve.
Likewise, the expression of MHC class II molecules can be
knocked out in order to reduce the risk of rejection of
the graft.
In yet another embodiment of the invention, the
three-dimensional culture system could be used in vitro
to produce biological products in high yield. For
example, a cell which naturally produces large quantities
of a particular biological product (e.q., a growth
factor, regulatory factor, peptide hormone, antibody,
etc.), or a host cell genetically engineered to produce a
foreign gene product, could be clonally expanded using
the three-dimensional culture system in vitro. If the
transformed cell excretes the gene product into the
nutrient medium, the product may be readily isolated from
the spent or conditioned medium using standard separation
technigues (e.q., HPLC, column chromatography,
electrophoretic techniques, to name but a few). A
"bioreactor~ has been devised which takes advantage of
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~ ~ ~ 8 ~ JS 95 111 39'
- I~EAIUS 26 NOV'96
the flow method for feeding the three-dimensional
cultures in vitro. Essentially, as fresh media is passed
through the three-dimensional culture, the gene product
is washed out of the culture along with the cells
released from the culture. The gene product is isolated
(e.q., by HPLC column chromatography, electrophoresis,
etc.) from the outflow of spent or conditioned media.
I.. EXAMPLE: THREE-DIMENSIONAL HEART
VALVE CULTURE SYSTEM
The three-dimensional culture of the present
invention provides for the growth of stromal cells such
as fibroblasts upon decellularized heart valves in vitro,
in a system designed to mimic physiologic conditions in
-~ vivo. Importantly, the cells replicated in this system
synthesize proteins similar to those produced by the
normal aortic wall and leaflet cells.
Heart valve extracellular matrix is composed mainly
of elastin and collagen types I and III. The following
example describes a method for growing transplantable or
bioprosthetic heart valve tissue in culture by
inoculating stromal cells from an exogenous source on
aortic walls and leaflets, and obtaining morphologically
and functionally normal human cells proliferating on the
three-dimensional framework.
-- A.... MATERIAL AND METHODS
1.. CELLS AND PORCINE TISSUE
Porcine aortic walls and leaflets were washed with
phosphate buffered saline and used fresh or after being
frozen at -20C to -70C in sterile water or after
detergent andtor enzyme extraction or any aforementioned
tissue in combination with sterilization techniques as
described in U.S. Patent No. 4,776,853. See Section 5.1.
Dermal foreskin fibroblasts were cultured in vitro
by routine procedures. Fibroblasts used in the studies
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AMENDED SHEE~
WO96108213 PCT~S95/11395
were in their eighth passage at the time of seeding to
the porcine tissues.
6 .1. 2 . FO~ WEER IN V~TRO C~LTURE8 OF
PORCINE AORTIC LEl'.FLET8 AND WALL8
Porcine aortic leaflets and walls were seeded in
eight well dishes with lx105 human dermal fibroblasts and
cultured for one day. The aortic walls and leaflets were
transferred into new well dishes and grown for an
additional four weeks. The eight cultures were made up
of: (l) previously frozen leaflet seeded with human
fibroblasts; (2) previously frozen wall seeded with human
fibroblasts; (3) previously frozen leaflet without
seeding; (4) previously frozen wall without seeding; (5)
fresh leaflet seeded with human fibroblasts; (6) fresh
wall-seeded with human fibroblasts; (7) fresh leaflet
without seeding; and (8) fresh wall without seeding. The
cultures were labeled with t35S]-methionine and [35S]-
cysteine (Tran 35S-Label, ICN) for four hours. The
samples were boiled in Laemmli sample buffer containing
~-mercaptoethanol, fractionated by SDS polyacrylamide gel
electrophoresis (SDS-PAGE), and analyzed by
autoradiography.
6.1.3. LES8 THAN 1 WEE~ TO 18-WEER
IN VITRO CULTURE8 OF POR~T~
AORTIC LEAFLET8 AND WAh~8
Decellularized porcine leaflet or wall tissues were
housed in a multi-well dish (one piece of tissue/well) as
described above in Section 6.l.2. The human dermal
fibroblasts were suspended in a nutrient-rich growth
medium and were seeded onto the specific types of porcine
leaflet or wall tissues such as: l) frozen leaflets and
walls; 2) electron beamed leaflets; 3) detergent and/or
enzyme extracted leaflets and walls; and 4) detergent
and/or enzyme extracted + electron beamed leaflets.
Each culture dish was maintained at 37C in a
sterile, static culture (no media flow) environment.
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WO96108213 ~ PCT~S95/1139
These human fibroblast-porcine tissue composites which
grow in the tissue culture dish are referred to as heart
valve constructs. The constructs were analyzed for:
a) Cell Distribution
In order to track the distribution and migration of
the human dermal fibroblasts into the porcine tissue
matrix, an antibody (human anti-prolyl-4-hydroxylase)
(DAK0-0-Fibroblast, 5B5, Code No. M 877) was used to
identify the cells post-colonization. The antibody does
not cross-react with porcine tissue. See Figure 5.
b) Cell Viability
i) MTT Assay: This assay is used to assess the
viability of cells after growing on the porcine matrix.
Metabolically active (living) fibroblasts convert the MTT
substrate (0.5mg/ml) into an insoluble purple precipitant
within the cells. The purple precipitant can be
visualized by the naked eye and this reflects the pattern
of the viable fibroblast distribution on the porcine
matrix. The MTT reaction can be quantified by measuring
the optical density with a spectrophotometer (540nm)
after extraction in isopropanol as described in Triglia,
D., et al., l99l, Toxic. in Vitro 5:573-578.
ii) Glucose consumption: As an indicator of
fibroblast viability, nutrient consumption (glucose) and
metabolic waste products (lactate) contained in the
tissue construct are measured as described in
Halberstadt, C.R., et al., 1994, Biotechnology and
Bioengineering 43:740-746. Viable fibroblasts decrease
the concentration of glucose over time and increase the
concentration of lactate.
c) Cell Prolifer~tion
i) Thymidine r~-thy~ IncorPor~tion: Radioactive
thymidine (lO ~Ci) is added to the nutrient-rich media
during the 24-72 hr culture of tissue constructs. When
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WO96/08213 PCT~S9S/11395
fibroblasts in the constructs divide to produce
additional cells, some of the 3H-thy becomes incorporated
in the DNA of the cells. Excess, non-incorporated 3H-thy
is removed after washing the labelled constructs in 1%
triton-X-lO0 for 2 hr and rinsing in PBS. The
incorporated 3H-thy can be measured using a scintillation
counter.
ii) Brd~ Incorporation: An alternative method for
measuring fibroblast proliferation in tissue constructs
is to add 5-bromodeoxyuridine (BrdU) to the culture
media. BrdU is a non-radioactive, thymidine analog which
incorporates into newly synthesized DNA of dividing
fibroblasts. Tissues are incubated in BrdU-containing
media for 24-48 hr. The fibroblasts containing BrdU can
be v1sualized in the histology sections of the tissue
constructs using a monoclonal antibody to the BrdU,
followed by an enzyme-chromogen detection system using
the Zymed Kit. (ZYMED Laboratories, Inc. San Francisco,
Ca).
d) Protein AssaYs
These methods utilize radiolabelled amino acids
which are added to the nutrient-rich media during the
tissue culture process. The radiolabelled amino acids
are incorporated into newly synthesized proteins in the
tissue constructs and can be measured using a
scintillation counter and/or extracted and separated on a
polyacrylamide (10%) gel by their molecular weights. The
gel is washed in salicylic acid (lM), then exposed to an
X-ray film (4-16 hr at 4 -25C) which, upon developing,
detects the images of radiolabelled proteins.
~ Proline Labelinq: Proline is a major amino
acid constituent of the collagen proteins in the tissue
constructs. The amount of radioactive proline
incorporated (incubation in lO~Ci/ml for 24-72 hr) is
quantified by scintillation counting to reflect the
amount of collagen being newly synthesized. ~xcess, non-
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2 ~ ~ 9 ~ ~ ~ PCT~S95111395
WO96/08213
incorporated label is removed after washing the labeled
constructs in 1% triton X-lO0. Ascorbic acid (25-
50~g/ml) can be used as a positive inducer of collagen
synthesis through activation of the propyl4-hydrox-ylase
enzyme.
ii) 358-Cysteine/Methionine Labelinq: Constructs
are incubated for 0.5 hr in medium free of cysteine and
methionine, then in medium containing labeled cysteine
and methionine (0.2mCi/ml) for 4 hr. The radioactive
isotopes are incorporated into newly synthesized
proteins. The labelled tissues can then be digested in
laemeli sample buffer under reducing (~-mercaptoethanol)
conditions and separated by SDS-PAGE. Specific proteins
can be quantified by western blotting.
e) Protein ImmunohistochemistrY
This method detects specific proteins in a
histological section of tissue using monoclonal or
polyclonal antibodies. The antibodies used specifically
detect human proteins and react with: l) human
fibroblasts (prolyl-4-hydroxylase); 2) a small component
of human elastin fibers (in valve wall and leaflet
tissue); and 3) human tenascin (matrix glycoprotein).
The antibody to the target protein is added to
deparaffined or frozen sections. A second antibody which
recognizes the primary antibody, is conjugated to an
enzyme-chromogen visualization system.
6 .1. 4 . IN VITRO COT Q~TZATION OF AORTIC WAI.I-S
AND l El~FLET8 lJNDER DYNAMIC CONDITIONS
Porcine leaflets were glued (medical grade
cyanoacrylate) either along one surface (immobilizing the
tissue) or on one edge (allowing some movement) to a
polycarbonate cassette sterilized by electron beam
radiation (E-beam). Human fibroblasts were seeded
dynamically (~ ml/min flow rate) on these tissues and
cultured for three days. The tissues were excised from
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WO96/08213 PCT~S95/11395
the cassette and labeled with [35S]-methionine and [35S]-
cysteine for four hours. Tissues were boiled in Laemmli
sample buffer, insoluble material was pelleted, and
supernatants were fractionated by SDS-PAGE and visualized
by autoradiography.
6.1.5. IN VITRO COLONIZATION OF WHOLE AORTIC
VALVES UNDER DYNAMIC CONDITION8
Whole porcine valves were either sterilized by E-
beam radiation or disinfected by an
antibiotic/antimycotic solution. Valves were placed in
E-beam sterilized "bioreactor" and seeded with 40-50 X106
cells at a flow rate of 15-50 ml/min in recirculating
nutrient-rich medium. After culture for up to 4 (or 8
weeks)with nutrient exchanges every week, the constructs
were evaluated by MTT assay, histological staining and
immunohistochemistry.
6.2. RESULTS
6.2.1. PORCINE AORTIC LEAFLET8 AND WALLS
While the frozen, thawed, unseeded aortic leaflets
and walls did not incorporate appreciable amounts of
label (Fig. 1, lanes 3 and 4, respectively), the leaflets
and walls which were seeded with human fibroblasts
incorporated the radioactive amino acid precursors (35S-
cys/met) and synthesized proteins ranging in molecular
weight from approximately 29,000 to 200,000 daltons (Fig.
1, lanes 1 and 2, respectively).
Lanes 5 through 8 describe corresponding results
obtained with fresh, unfrozen leaflets and walls.
Seeding fresh, unfrozen aortic leaflets and walls with
human fibroblasts, resulted in an increase in the
incorporation of the amount of radioactivity, in lanes 5
and 6. This increase is similar to that observed in
lanes 1 and 2, respectively. Lanes 7 and 8, containing
unseeded fresh, unfrozen aortic leaflets and walls,
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WO96/08213 ~ PCT~S95/11395
respectively, demonstrated protein synthesis by
endogenous viable cells. The aortic walls were less
metabolically active than the leaflets. Of particular
interest is the fact that the protein profile shown in
lane 7 is similar to the protein profile in lane l,
indicating that proteins synthesized by fibroblasts
seeded onto frozen porcine leaflets are similar to
proteins that are synthesized by the endogenous cells of
normal, fresh porcine leaflets.
6.2.2. COLONIZATION OF AORTIC WALL8
AND LEAFLET8 AT 8-18 WEER8
Human dermal fibroblasts were able to colonize every
tissue type from biopsies of aortic leaflets and walls
over all time intervals described in Table II. The
greatest fibroblast penetration of the porcine matrix
occurred in leaflets, specifically in detergent and/or
enzyme extracted leaflets (Figure 2). Overall, the cell
distribution in the detergent and/or enzyme extracted
leaflets cultured 8 to 18 weeks appeared to approach cell
densities typical of a fresh porcine leaflet. Figure 3
represents cell distribution in detergent and/or enzyme
extracted leaflets cultured for 4 weeks.
Cell viability assessments (MTT assay) demonstrated
that the human fibroblasts remained metabolically alive
even after 18 weeks. The fibroblasts were also shown to
be proliferating (3H-thy incorporation assay) throughout
the culture process (Figure 4).
Protein production, measured as collagen synthesis
(3H-proline labeling) indicated that the human dermal
fibroblasts were producing collagen and some proteins
that are present in porcine leaflets (35S-
cysteine/methionine labeling) (Figure 5).
Proteins typical of heart valve tissue were
identified by immunohistochemistry using specific
antibodies. Fibroblasts produced human tenascin to
supplement the existing porcine scaffolding (Figure 6).
WO96/08213 ~ PCT~S95111395
6.2.3. COLONIZATION OF AORTIC WALL8 AND
LEAFLETS ~NDER DYNAMIC CONDITIONS
Lanes 1 and 2 (Fig. 7) containing samples of porcine
leaflets that were glued along their entire surface, and
cultured under dynamic flow had no appreciable staining.
Lanes 3 and 4 show porcine leaflets that were glued on
one edge, with an appreciable amount of radioactivity was
incorporated after growth for three days post seeding;
when porcine leaflets were glued along an entire surface,
minimal [35S] was incorporated into protein. An unseeded,
E-beam sterilized leaflet used as a control (lane 5)
showed no incorporation of radioactivity.
Thus, porcine aortic leaflets and walls can be
statically or dynamically seeded with human fibroblasts.
These human fibroblasts attach and colonize the aortic
leaflet and wall scaffolds, and remain metabolically
active by secreting extracellular matrix molecules.
6.2.4. IN VITRO COLONIZATION OF WHOLE AORTIC
VALVES UNDER DYNAMIC CONDITIONS
Human fibroblasts grew under dynamic conditions, on
a porcine matrix which was previously decellularized by
detergent and/or enzyme treatment. The proliferating
cells were labeled with Brdu and detected using an
antibody to Brdu. See Figure 8.
When the human fibroblasts were grown on a porcine
matrix which was previously decellularized by detergent
and/or enzyme treatment + electron beamed, the matrix
seeded under dynamic, pulstile flow conditions had
greater and more uniform fibroblast attachment that the
matrix grown under static conditions, as shown by using
the MTT assay (an indicator of cell viability as
described in Section 6.1.3 (b) above). See Figure 9.
The present invention is not to be limited in scope
by the specific embodiments described which are intended
as single illustrations of individual aspects of the
invention, and functionally equivalent methods and
- 45 -
WO96/08213 ~ 8 ~ ~ PCT~S95/11395
components are within the scope of the invention, in
addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such
modifications are intended to fall within the scope of
the appended claims.
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